Abstract:

A freezing microtome for the production of microscopable thin sections of
tissue samples (60), comprising a cooling device (24, 34, 36, 38, 40, 42,
46, 48, 50, 52) for freezing the tissue samples (60), a cutting device
for cutting the frozen tissue samples (60) and a working chamber, in
which the cutting device and at least a part of the cooling device (24,
34, 36, 38, 40, 42, 46, 48, 50, 52) are arranged is described. The part
of the cooling device (24, 34, 36, 38, 40, 42, 46, 48, 50, 52) arranged
in the working chamber contains a liquid coolant (70), in which the
tissue samples (60) can be inserted for freezing.

Claims:

1. A freezing microtome for the production of microscopable thin sections
of tissue samples, comprising:a cooling device for freezing the tissue
samples (60);a cutting device for cutting the frozen tissue samples;a
working chamber in which the cutting device and at least a part of the
cooling device are arranged, wherein the cooling device includes a tank
arranged in the working chamber for receiving a liquid coolant into which
the tissue samples are insertable for freezing the tissue samples; anda
rinsing device arranged in the working chamber (20) for rinsing the
frozen tissue samples with a cooled rinsing liquid.

2. The freezing microtome according to claim 1, wherein the liquid coolant
has a temperature within a range from approximately -60.degree. C. to
0.degree. C. for freezing the tissue samples.

3. The freezing microtome according to claim 1, wherein the liquid coolant
has a specific heat capacity within a range from 1.6 to 3.5 kJ/kgK.

4. The freezing microtome according to claim 1, wherein the cooling device
includes a compression refrigerating machine for cooling the liquid
coolant.

5. The freezing microtome according to claim 4, wherein the compression
refrigerating machine includes an evaporator arranged in the tank.

6. The freezing microtome according to claim 5, wherein the compression
refrigerating machine includes a further evaporator arranged in the
working chamber outside of the tank.

7. The freezing microtome according to claim 4, further comprising a
cabinet casing including an upper part and a lower part, wherein the
working chamber is arranged in the upper part of the cabinet casing, and
wherein the compression refrigerating machine includes a compressor
arranged in the lower part of the cabinet casing.

8. The freezing microtome according claim 1, wherein the cooling device
includes a flow-through device having a reservoir for storing the liquid
coolant, a cooling aggregate for cooling the coolant stored in the
reservoir, and a circulation pump which delivers the liquid coolant via
an inflow from the reservoir into the tank and via an outflow from the
tank back into the reservoir.

9. The freezing microtome according to claim 1, wherein the rinsing device
includes a rinsing tank which contains the cooled rinsing liquid and is
arranged near the part of the cooling device arranged in the working
chamber.

10. The freezing microtome according to claim 9, wherein the temperature
of the rinsing liquid is adjusted to a temperature which is approximately
equal to the temperature of the liquid coolant.

11. A method for producing microscopable thin sections, comprising the
following steps:providing a freezing microtome having a liquid coolant, a
cooled rinsing liquid, and a cutting device;inserting a tissue sample
into the liquid coolant to freeze the tissue sample;rinsing the tissue
sample with the cooled rinsing liquid; andcutting the frozen tissue
sample with the cutting device after the tissue sample has been rinsed
with the cooled rinsing liquid.

12. The method according to claim 11, wherein the tissue sample is wrapped
in a protective cover before being inserted into the liquid coolant.

13. The method according to claim 12, wherein the tissue sample is cut
together with the protective cover.

14. The method according to claim 11, wherein the tissue sample is
attached to a mounting device before being inserted into the liquid
coolant and is inserted into the liquid coolant together with the
mounting device and removed from the liquid coolant after freezing.

15. The method according to claim 11, wherein the temperature of the
liquid coolant for freezing the tissue sample is selected within a range
from approximately -60.degree. C. to 0.degree. C.

16. The method according to claim 11, wherein the specific heat capacity
of the liquid coolant is selected within a range from approximately 1.6
to 3.5 kJ/kgK.

[0002]The invention relates to a freezing microtome and a method for the
production of microscopable thin sections of tissue samples. In
particular, the invention relates to a freezing microtome comprising a
cooling device for freezing the tissue samples, a cutting device for
cutting the frozen tissue samples and a working chamber, in which the
cutting device and at least a part of the cooling device are arranged.

BACKGROUND OF THE INVENTION

[0003]By means of freezing microtomes of the aforementioned type thin
section specimens are produced from tissue samples, which are
subsequently observed under a microscope for diagnostic purposes. For
this, the tissue samples are firstly frozen in a cooling device, also
referred to as cryostat, and then cut by means of a cutting device. The
cooling device and the cutting device are arranged together in a working
chamber of the freezing microtome.

[0004]The cooling device usually comprises freezing ribs cooled by means
of Peltier elements, to which slides holding the tissue samples are
attached. This type of sample cooling has the disadvantage that during
the freezing process elongated ice crystals are formed in the tissue due
to the crystallization of the cellular and extracellular water, which
perforate the cells and united cell structures of the tissue and affect
the histomorphology adversely. Such tissue damages are also referred to
as freezing artifacts. A possible adverse affect by freezing artifacts
consists e.g. of the damaged cells releasing endogenous enzymes, which
become active during an RNA preparation and thus degrade the substrate
used in the preparation. This reduces the sample quality and as a
consequence makes a reliable diagnosis more difficult.

[0005]To prevent freezing artifacts, it is alternatively suggested to
freeze the tissue samples externally, i.e. outside of a microtome,
directly by means of liquid nitrogen or by using nitrogen-cooled
isopentane. However, the use of liquid nitrogen is complex and expensive.
Further, freezing of the tissue samples outside of the microtome involves
the risk to mix up the tissue samples.

[0006]Regarding prior art, it is further referred to U.S. Pat. No.
6,615,592 B2. Therein a cooling device is described, wherein a liquid
coolant is led over the tissue samples to freeze them.

SUMMARY OF THE INVENTION

[0007]It is the object of the invention to provide a freezing microtome of
the aforementioned type as well as a method for the production of
microscopable thin sections of tissue samples which allow for an easy
handling of the tissue samples while largely avoiding freezing artifacts.

[0008]The invention solves this object with respect to the freezing
microtome by the subject-matter of claim 1.

[0009]The invention provides the integration of a liquid coolant, which
according to latest findings allows a freezing of tissue samples while
largely avoiding freezing artifacts, in a freezing microtome. As both the
liquid coolant and the cutting device are arranged in the working chamber
of the freezing microtome, an easy handling of the tissue samples for the
thin section preparation is guaranteed.

[0010]For freezing the tissue samples, the liquid coolant preferably has a
temperature within a range from approximately -60° C. to 0°
C. Further, it preferably has a specific heat capacity within a range
from approximately 1.6 to 3.5 kJ/kgK. Thus it has turned out that if a
liquid coolant with these characteristics is used, the cellular and
extracellular water when the tissue is frozen only forms small, spherical
ice crystals which neither damage the cells nor the cell walls. Thus the
native histomorphology remains largely preserved during freezing. This
preservation of the morphology facilitates the so-called instantaneous
section diagnosis for the pathologist. As the cells and united cell
structures are largely preserved and thus the aforementioned endogenous,
degrading enzymes are virtually not released, e.g. also very unstable
mRNA molecules can be extracted in relative high concentrations. As these
information molecules gain in importance regarding the diagnosis of e.g.
cancer, the use of a liquid coolant of the aforementioned type promises a
significant progress regarding the diagnosis.

[0011]Preferably, the cooling device includes a tank arranged in the
working chamber for receiving the liquid coolant and the tissue samples.
The tank arranged in the working chamber allows for various embodiments
of the inventive freezing microtome. In a particularly simple embodiment
the liquid coolant is e.g. cooled externally, i.e. outside of the
microtome, and then inserted into the tank. In order to simplify the
handling it is however advantageous, if the coolant is cooled in the
freezing microtome itself.

[0012]For this, the cooling device can e.g. include a compression
refrigerating machine for cooling the liquid coolant, which works with
compression and expansion elements as well as with heat exchangers to
implement a thermodynamic cycle.

[0013]Preferably, the compression refrigerating machine includes an
evaporator arranged in the tank. This evaporator is e.g. formed from a
tube, in which a refrigerant such as e.g. R404a evaporates and thus
extracts warmth from the liquid coolant, which surrounds the tube in the
tank.

[0014]In a further advantageous embodiment the compression refrigerating
machine includes at least a further evaporator, which is arranged in the
working chamber outside of the tank. This further evaporator can e.g. be
arranged such that it provides for an air cooling in the working chamber.

[0015]The freezing microtome can also be designed such that the cooling
device includes a flow-through device having a reservoir for storing the
liquid coolant, a cooling aggregate for cooling the coolant stored in the
reservoir and a circulation pump, which delivers the liquid coolant via
an inflow from the reservoir into the tank and via an outflow from the
tank back into the reservoir. This flow-through device can be provided
alternatively or additionally to the aforementioned compression
refrigerating machine. In the latter case the cooling aggregate causes
e.g. a precooling of the coolant stored in the reservoir, while the
compression refrigerating machine provides for a cooling of the coolant
in the tank.

[0016]The freezing microtome has a rinsing device arranged in the working
chamber for rinsing the tissue samples frozen by means of the liquid
coolant with a cooled rinsing liquid. If the tissue samples are wrapped
in protective covers to prevent cross-contaminations of the tissue
samples via the coolant, the coolant adhering to the protective covers
can be removed by means of the rinsing device before the tissue samples
are supplied to the cutting device together with the protective covers.

[0017]Preferably, the rinsing device includes a rinsing tank containing
the rinsing liquid, which is arranged near the part of the cooling device
arranged in the working chamber. By this arrangement of the rinsing tank
a contamination of the cutting device by the coolant adhering to the
protective covers of the tissue samples can be prevented. As rinsing
liquid e.g. diluted or undiluted ethanol or isopropanol can be used.

[0018]Further it is advantageous to cool the rinsing liquid as well. For
this, in the working chamber a distinct cooling device associated with
the rinsing device can be provided. Alternatively, the cooling device
determined for cooling the liquid coolant can also be used for cooling
the rinsing liquid. When using a compression refrigerating machine, this
can e.g. be caused by an additional evaporator arranged in the rinsing
tank.

[0019]Preferably, the cooled rinsing liquid has a temperature which is
approximately equal to the temperature of the liquid coolant. Thus it is
assured that the tissue samples also still during the rinsing process
keep a temperature, which proved to be optimal with respect to the
prevention of freezing artifacts. In particular, the tissue samples do
not thaw during rinsing, whereby cutting the tissue samples would be
impeded.

[0020]According to a further aspect of the invention a method for the
production of microscopable thin sections of tissue samples according to
claim 11 is provided.

[0021]Preferably, the tissue samples are respectively wrapped in a
protective cover before being inserted into the liquid coolant. As
protective cover e.g. a plastic hose can be used. The protective cover
prevents that the coolant comes into direct contact with the tissue
sample. Cross contaminations via the liquid coolant can thus be
prevented.

[0022]The tissue samples can be cut together with the protective covers,
in which they have been wrapped before being inserted into the liquid
coolant. This facilitates the implementation of the method.

[0023]The implementation of the method is further facilitated in that the
respective tissue sample is attached to a mounting device before being
inserted into the liquid coolant and inserted into the liquid coolant and
removed from the liquid coolant after freezing together with the mounting
device.

BRIEF DESCRIPTION OF THE DRAWING VIEWS

[0024]The invention will be explained in more detail in the following on
the basis of embodiments with reference to the Figures, wherein:

[0025]FIG. 1 shows a perspective view of an inventive freezing microtome
according to a first embodiment;

[0026]FIG. 2 shows a perspective view of the freezing microtome according
to FIG. 1, wherein parts of the freezing microtome are omitted;

[0027]FIG. 3 shows a schematic illustration of a refrigeration cycle
provided in the freezing microtome according to FIGS. 1 and 2;

[0028]FIG. 4 shows a perspective view of an inventive freezing microtome
according to a second embodiment, wherein parts of the freezing microtome
are omitted;

[0029]FIG. 5 shows a top view of the freezing microtome according to FIG.
4; and

[0030]FIGS. 6A, 6B and 6C show schematic illustrations showing how a
tissue sample is wrapped in and inserted into the liquid coolant.

DETAILED DESCRIPTION OF THE INVENTION

[0031]FIG. 1 shows a freezing microtome 10 according to a first
embodiment. The freezing microtome 10 has a cabinet casing 12, on which a
console 14 is mounted. Control panels 16 and 18 with buttons and display
elements are on the console 14, via which buttons and display elements an
operator can control the operation of the freezing microtome 10.

[0032]The freezing microtome 10 has a working chamber 20 with a movable
cover 22. In FIG. 1 the cover 22 is inserted and thus the working chamber
20 is open. In the working chamber 20 a tank 24 and a cutting device 26
are arranged. When the freezing microtome 10 operates, the tank 24
contains a liquid coolant not shown in FIG. 1. Tissue samples are
inserted into the coolant, the temperature of which is within a range
from approximately -60° C. to 0° C. By means of the coolant
the tissue samples are frozen and thus hardened. Then, from the hardened
tissue samples thin sections can be produced by means of the cutting
device 26.

[0033]The cutting device 26 comprises a clamping device 28 and a knife
facing the clamping device 28, which is not shown in FIG. 1. A slide,
which holds a tissue sample, can be attached to the clamping device 28.
The clamping device 28 can be approached to the knife facing it. If the
clamping device 28 is approached to the knife, thin sections can be
planed off from the frozen tissue sample by moving the clamping device 28
upward and downward together with the tissue sample. This upward and
downward movement of the clamping device 28 can be caused manually via a
handwheel 30 or motor-driven.

[0034]In FIG. 1, further a collection tank 32 for condensate is shown,
which is arranged at the front side of the cabinet casing 12.

[0035]In FIG. 2, again the freezing microtome 10 according to FIG. 1 is
shown.

[0036]Whereby in FIG. 2 some parts of the freezing microtome 10, in
particular the side walls of the cabinet casing 12 and the console 14 are
omitted to show parts of a cooling device, which is used in the first
embodiment.

[0037]This cooling device comprises apart from the tank 22 shown in FIG. 1
inter alia a compressor 34, a collection dryer 36, a condenser 38, a
tubular evaporator 40, a lamella evaporator 42 and a wall evaporator,
which is arranged in a side wall 44 of the working chamber 20, e.g.
foamed in place, and therefore cannot be seen in FIG. 2.

[0038]The compressor 34, the collection dryer 36 and the condenser 38 are
located in the lower part of the cabinet casing 12 below the working
chamber 20. The tubular evaporator 40 is arranged in the tank 24. The
lamella evaporator 42 is arranged in the working chamber 20 outside of
the tank 24. The cooling device comprises further components such as
tubes, temperature sensors and expansion valves, which are not further
identified in FIG. 2.

[0039]With reference to the schematic illustration according to FIG. 3, in
the following the refrigeration cycle of the cooling device used in the
freezing microtome 10 is described.

[0040]The cooling device works with a refrigerant such as e.g. R404a. The
pressure ratios are selected in the refrigeration cycle such that the
evaporating temperature of the refrigerant in this embodiment is between
approximately -40° C. to -50° C.

[0041]In the compressor 34 the refrigerant present in the vaporous state
of aggregation is compressed and thus heated up. The hot refrigerant
vapor is then cooled down in the condenser 38 so far that it condenses.
Subsequently, the liquid refrigerant flows through the collection dryer
36. The collection dryer 36 has on the one hand the function to receive
and store the liquid refrigerant. On the other hand, it has the function
to dehumidify the liquid refrigerant to prevent icing of various
components of the refrigeration cycle, e.g. of the tubular evaporator 40
and the lamella evaporator 42.

[0042]The liquid refrigerant then flows on the one hand via a first
expansion valve 46 into the lamella evaporator 42 arranged in the working
chamber 20 outside of the tank 24 and on the other hand via a second
expansion valve 48 into the tubular evaporator 40 arranged in the tank 24
and into the wall evaporator arranged in the side wall 44 of the working
chamber 20, which wall evaporator is referred to with 50 in FIG. 3. In
the branch of the refrigeration cycle, wherein the lamella evaporator 42
is located, a first overheating sensor 52 is provided. Correspondingly, a
second overheating sensor 54 is provided in the branch of the
refrigeration cycle, wherein the tubular evaporator 40 and the wall
evaporator 50 are arranged.

[0043]The expansion valves 46 and 48 reduce the pressure of the liquid
refrigerant flowing therethrough. In the evaporators 40, 42 and 50 the
refrigerant evaporating due to the pressure drop extracts evaporation
heat from the medium, in which the respective vaporizer 40, 42 or 50 is
located. In this manner, the liquid coolant, which surrounds the tubular
evaporator 40 in the tank 24 as well as the air, which surrounds the
lamella evaporator 42 and the wall evaporator 50 in the working chamber
20, are cooled down.

[0044]Eventually, the refrigerant vapor is sucked in by the compressor 34
and again compressed, whereupon the refrigeration cycle starts anew.

[0045]In FIG. 4, a second embodiment is shown, wherein a rinsing device is
additionally provided in the working chamber 20. Apart from this rinsing
device the second embodiment is identical to the first embodiment.

[0046]In the second embodiment the rinsing device is simply designed as
rinsing tank 56. A rinsing liquid, e.g. ethanol, can be filled into the
rinsing tank 56, in which the tissue samples, after they have been frozen
in the liquid coolant and have been taken out of the tank 24, can be
purified.

[0047]As the top view according to FIG. 5 also shows, the rinsing tank 56
is arranged in the working chamber 20 between the tank 24 and the cutting
device 26. In particular, the rinsing tank 56 is located in immediate
proximity of the tank 24. Thus it is assured that the working chamber 20
is not contaminated with the liquid coolant, if the operator takes the
frozen tissue samples out of the tank 24.

[0048]Preferably, the rinsing liquid contained in the rinsing tank 56 is
cooled. This can e.g. be caused by a further evaporator, not shown in
FIGS. 4 and 5. Such an evaporator could e.g. be designed according to the
tubular evaporator 40 arranged in the tank 24. The temperature to which
the rinsing liquid is cooled, is in this embodiment approximately
identical to the temperature of the liquid coolant.

[0049]In the above described embodiments the liquid coolant is simply
filled into the tank 24. However, this simple design is to be understood
only as an example. Thus, it is e.g. also possible to transfer the liquid
coolant in a cycle from a liquid reservoir, which is located inside the
cabinet casing 12, via an inflow into the tank 24 and via an outflow from
the tank 24 back into the liquid reservoir. To this end, a circulation
pump can be provided in the freezing microtome 10 which pumps the liquid
coolant into the tank 24 and out of the tank 24.

[0050]A corresponding cycle can also be provided for the rinsing liquid
contained in the rinsing tank 56. In this case, a liquid reservoir for
the rinsing liquid, a circulation pump as well as an inflow and an
outflow for the rinsing tank 56 are to be provided in the freezing
microtome 10.

[0051]With reference to FIGS. 6A, 6B and 6C in the following it shall be
illustrated by way of example, how microscopable thin sections of tissue
samples are produced by means of the freezing microtome 10.

[0052]As shown in FIG. 6A, first a tissue sample identified with 60 is put
into a protective cover 62, which is simply designed as plastic hose in
the present embodiment. The protective cover 62 is clamped in a mounting
device 64 shown in FIG. 6B together with the tissue sample 60 contained
therein. Thereby the protective cover 62 can e.g. be clamped such that
the ends thereof being open at first are pressed together and thus closed
by clamping arms 66, 68 arranged pairwise respectively, which are formed
at the mounting device 64 and of which in the side views according to
FIGS. 6B and 6C only respectively one clamping arm 66 or 68 is shown. In
FIGS. 6B and 6C this is only schematically indicated.

[0053]The mounting device 64 is then placed in the tank 24 together with
the protective cover 62 held thereon, in which the liquid coolant
indicated with 70 in FIG. 6C is located.

[0054]In the present embodiment the mounting device 64 is designed such
that it can simply be placed in the tank 24 and removed therefrom by the
operator. For this, the mounting device 64 has feet 72, with which it is
placed on the bottom of the tank 24. Further the mounting device 64 has
handholds 74, which project from the liquid coolant 70, when the mounting
device 64 is placed in the tank 24. The operator can thus simply take the
mounting device 64 out of the liquid coolant 70 at the handholds 74.

[0055]After the tissue sample 60 has been frozen in the liquid coolant 70,
it is supplied to the cutting device 26 together with the mounting device
64 to produce the desired thin sections.